Haemophilus ducreyi is the causative agent of chancroid, a sexually transmitted genital ulcerative disease. The pathogenic mechanisms responsible for ulcer formation and immunity to H. ducreyi infection are poorly understood. We previously characterized many of the genes encoding glycosyltransferases responsible for synthesis of the major glycoforms of H. ducreyi lipooligosaccharide (LOS). One of the genes identified in these studies is the LOS sialyltransferase. We recently extended our characterization of sialic acid metabolism in H. ducreyi by designing and executing a genetic screen that identified genes encoding the sialic acid transporter. In addition to identifying the transporter, we observed that H. ducreyi produces a non-LOS glycoconjugate containing terminal Gal-?1-4-GlcNAc. Western blot data suggests that this terminal Gal-GlcNAc disaccharide decorates a protein. Although much has been learned about Gram-negative glycoproteins in the last several years, it is apparent that we have just begun to understand the mechanisms of synthesis of these glycoconjugates and the roles that they play in the biology of pathogenic organisms and bacterial-host interactions. [unreadable] In this proposal, we describe experiments designed to identify the H. ducreyi glycoprotein, identify the composition of the complete carbohydrate decorating the protein and identify the genes encoding the enzymes responsible for glycosylation of this protein. These studies will form the basis for an R01 application that will examine, in depth, the structure, biosynthesis and function of the carbohydrate decoration on the H. ducreyi protein. Studies will also be proposed in that R01 application to determine the role of the protein in virulence using both in vitro models as well as the human challenge model developed in Stanley Spinola's laboratory. [unreadable] Identification of the protein will likely provide additional insight into the poorly understood pathogensis of chancroid, will provide new data that will contribute to our increasing understanding of bacterial glycoproteins and the role they play in bacterial pathogenesis and immunity, and may also provide insight into the development of novel strategies for prevention of H. ducreyi infection. [unreadable] [unreadable] [unreadable]